The overall goal of this work is to develop anatomical, functional, and molecular magnetic resonance imaging (MRI) techniques that allow non-invasive assessment of brain function and apply these tools to study plasticity and learning in the rodent brain. MRI techniques are having a broad impact on understanding brain. Anatomical based MRI has been very useful for separating gray and white matter and detecting numerous brain disorders. Functional MRI techniques enable detection of regions of the brain that are active during a task. Molecular MRI is an emerging area, whose major goal is to image a large variety of processes in tissues. The goal of this project is to translate MRI developments in all these areas to study system level changes that occur in the rodent brain during plasticity and learning. Aim 1: Over the past few years, we have completed studies in the rodent brain that acquired very high temporal and spatial resolution functional MRI (fMRI) to monitor changes in hemodynamics as a surrogate marker of electrical activity during forepaw stimulation. Over the past couple of years we have demonstrated that fMRI from single venuoles can be detected with BOLD fMRI. This year we have published that single arterioles from deep cortex can be effectively imaged using blood volume based MRI techniques. Consistent with our understanding arteriole blood volume responds earlier that venous BOLD and the two vessel populations can be separated based on time to peak response. In blood volume the arteriole contribution dominates as compared to the tissue or venuole contributions. For BOLD the venuole contribution dominates compared to the arteriole contribution. We are verifying our MRI assignment of vessels and determining what fraction of arterioles and venuoles can be detected. Over the past couple of years we have demonstrated that the onset of fMRI enables extracting information about the onset of neural activity in a brain region. Over the past year we have accumulated data to asses the determinants of the early hemodynamic response and in particular the role of pericytes in initiating the early response. An alternative hypothesis that more co-ordinated cellular activity occurs is also being pursued. Aim 2: Over the past several years we have demonstrated that manganese chloride enables MRI contrast that defines neural architecture, can monitor activity, and can be used to trace neural connections. Over the last couple of years we have completed the assignment of cortical layers detected using manganese enhanced MRI by comparison to histology and have demonstrated that functional anatomy of several cortical regions of the rodent brain can be defined in individual animals. We have completed a study that shows that detecting olfactory bulb layers is a sensitive way to monitor neurodegeneraton in a mouse model of app induced olfactory bulb degeneration. This demonstrates the MRI at this very high level of resolution can detect layer specific degeneration. A hippocampal slice preparation has been established to study the synaptic mechanism underlying the MRI properties of manganese. Results indicate that a number of transport processes influence the accumulation and transport of Mn in these slice cultures. This indicates that the slice cultures in combination with MRI will be a very useful platform for understanding the basis for MRI contrast. Finally, we have continues a collaboration with Dorian McGavern, where we have shown that Mn can enter the brain through the skull at specific locations and once inside the brain trace appropriate neural connections. We are completing a study that characterizes the major pathways that Mn can enter the brain via specific pathways through the skull. This opens the very exciting possibility of delivering Mn to specific locations in the brain non-invasively and possible extending these ideas to the delivery of drugs. Aim 3: Over the past few years we established a rodent model that uses peripheral denervation to study brain plasticity in response to the injury. Over the past couple of years we have shown that denervation of the infraorbital nerve leads to large increases in barrel cortex responses along the spared whisker pathway as well as large ipsilateral cortical activity consistent with our previous work in the forepaw and hindpaw. fMRI and manganese enhanced MRI predicted a strengthening of thalamo-cortical input along the spared pathway which was verified in slice electrophysiology studies in collaboration with John Isaac. Prior to this it was widely believed that the thalamo-cortical input was not capable of strengthening after the critical period. The mechanisms of this strengthening are under study. We have finished a long term study that has conclusively shown that the denervation is causing a re-activation of the ability of this synapse to demonstrate long term potentiation. This LTP is occurring via activation of silent synapses. Interestingly, LTP is not dependent on the NR2B subunit, but infusion of NR2B inhibitors into the rodent cortex indicate that NR2B is important for establishing silent synapses. We have shown that trimming whiskers has a similar effect as infraorbital nerve cut showing that down regulation of use is enough to cause this LTP in the good whisker pathway. Evidence from manganese based track tracing shows a strengthening of input into layer 2/3 and 5 from the contralateral cortex to the ipsilateral cortex from the healthy whiskers. We have demonstrated that we can use optogenetic stimulation to study this input and have shown that the mouse has similar plasticity to the rat. Over the past year we have demonstrated a very large up regulation of activity in layer five from the callosal inputs. We are studying the synaptic basis for these effects. In this case this is a synapse that retains the ability for LTP and so we expect the basis for the strengthening is via an LTP like mechanism. Aim 4: Progress in this aim over the past year has been slow due to lack of a specific person to carry out the work. Fortunately a new member of the laboratory has reactivated this exciting project. We will continue to develop awake fMRI tools to study learning in a model fo fear conditioning. We will validate that earlier Mn tracing experiments that indicated plasticity at novel synapses. And we will take the same route as with the infraorbital nerve cut and determine the synaptic basis for the changes using slice electrophysiology.